{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,29]],"date-time":"2026-10-29T11:07:16Z","timestamp":1793272036950,"version":"4.5.0"},"reference-count":55,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2023,3,13]],"date-time":"2023-03-13T00:00:00Z","timestamp":1678665600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"GLAdiator Biosciences Inc."},{"name":"MSU"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vaccines"],"abstract":"<jats:p>Under physiological conditions, phosphatidylserine (PS) predominantly localizes to the cytosolic leaflet of the plasma membrane of cells. During apoptosis, PS is exposed on the cell surface and serves as an \u201ceat-me\u201d signal for macrophages to prevent releasing self-immunogenic cellular components from dying cells which could potentially lead to autoimmunity. However, increasing evidence indicates that viable cells can also expose PS on their surface. Interestingly, tumor cell-derived extracellular vesicles (EVs) externalize PS. Recent studies have proposed PS-exposing EVs as a potential biomarker for the early detection of cancer and other diseases. However, there are confounding results regarding subtypes of PS-positive EVs, and knowledge of PS exposure on the EV surface requires further elucidation. In this study, we enriched small EVs (sEVs) and medium\/large EVs (m\/lEVs) from conditioned media of breast cancer cells (MDA-MB-231, MDA-MB-468) and non-cancerous cells (keratinocytes, fibroblasts). Since several PS-binding molecules are available to date, we compared recombinant proteins of annexin A5 and the carboxylated glutamic acid domain of Protein S (GlaS), also specific for PS, to detect PS-exposing EVs. Firstly, PS externalization in each EV fraction was analyzed using a bead-based EV assay, which combines EV capture using microbeads and analysis of PS-exposing EVs by flow cytometry. The bulk EV assay showed higher PS externalization in m\/lEVs derived from MDA-MB-468 cells but not from MDA-MB-231 cells, while higher binding of GlaS was also observed in m\/lEVs from fibroblasts. Second, using single EV flow cytometry, PS externalization was also analyzed on individual sEVs and m\/lEVs. Significantly higher PS externalization was detected in m\/lEVs (annexin A1+) derived from cancer cells compared to m\/lEVs (annexin A1+) from non-cancerous cells. These results emphasize the significance of PS-exposing m\/lEVs (annexin A1+) as an undervalued EV subtype for early cancer detection and provide a better understanding of PS externalization in disease-associated EV subtypes.<\/jats:p>","DOI":"10.3390\/vaccines11030639","type":"journal-article","created":{"date-parts":[[2023,3,14]],"date-time":"2023-03-14T03:29:16Z","timestamp":1678764556000},"page":"639","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":32,"title":["Phosphatidylserine-Exposing Annexin A1-Positive Extracellular Vesicles: Potential Cancer Biomarkers"],"prefix":"10.3390","volume":"11","author":[{"given":"Gloria I.","family":"Perez","sequence":"first","affiliation":[{"name":"Institute for Quantitative Health Science and Engineering (IQ), Michigan State University, East Lansing, MI 48824, USA"},{"name":"College of Osteopathic Medicine, Michigan State University, East Lansing, MI 48824, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5950-3580","authenticated-orcid":false,"given":"Matthew P.","family":"Bernard","sequence":"additional","affiliation":[{"name":"Institute for Quantitative Health Science and Engineering (IQ), Michigan State University, East Lansing, MI 48824, USA"},{"name":"Department of Pharmacology & Toxicology, Michigan State University, East Lansing, MI 48824, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel","family":"Vocelle","sequence":"additional","affiliation":[{"name":"Department of Pharmacology & Toxicology, Michigan State University, East Lansing, MI 48824, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ahmed A.","family":"Zarea","sequence":"additional","affiliation":[{"name":"Institute for Quantitative Health Science and Engineering (IQ), Michigan State University, East Lansing, MI 48824, USA"},{"name":"Cell and Molecular Biology Program, Michigan State University, East Lansing, MI 48824, USA"},{"name":"College of Natural Science, Michigan State University, East Lansing, MI 48824, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Najla A.","family":"Saleh","sequence":"additional","affiliation":[{"name":"Institute for Quantitative Health Science and Engineering (IQ), Michigan State University, East Lansing, MI 48824, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthew A.","family":"Gagea","sequence":"additional","affiliation":[{"name":"Institute for Quantitative Health Science and Engineering (IQ), Michigan State University, East Lansing, MI 48824, USA"},{"name":"Lyman Briggs College, Michigan State University, East Lansing, MI 48824, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Doug","family":"Schneider","sequence":"additional","affiliation":[{"name":"GLAdiator Biosciences, Mill Valley, CA 94941, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maxine","family":"Bauzon","sequence":"additional","affiliation":[{"name":"GLAdiator Biosciences, Mill Valley, CA 94941, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Terry","family":"Hermiston","sequence":"additional","affiliation":[{"name":"GLAdiator Biosciences, Mill Valley, CA 94941, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9025-9582","authenticated-orcid":false,"given":"Masamitsu","family":"Kanada","sequence":"additional","affiliation":[{"name":"Institute for Quantitative Health Science and Engineering (IQ), Michigan State University, East Lansing, MI 48824, USA"},{"name":"Department of Pharmacology & Toxicology, Michigan State University, East Lansing, MI 48824, USA"},{"name":"College of Human Medicine, Michigan State University, East Lansing, MI 48824, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,3,13]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"639","DOI":"10.1016\/j.tcb.2015.08.003","article-title":"An apoptotic \u2018eat me\u2019signal: Phosphatidylserine exposure","volume":"25","author":"Segawa","year":"2015","journal-title":"Trends Cell Biol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"459","DOI":"10.1084\/jem.20040342","article-title":"Masking of phosphatidylserine inhibits apoptotic cell engulfment and induces autoantibody production in mice","volume":"200","author":"Asano","year":"2004","journal-title":"J. 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